The major goal of this project is to understand specific mechanisms by which RNA viruses reach evolutionary stasis or are compelled to rapid evolution (or extinction). We will use vesicular stomatitis virus populations as a model. We will do careful quantitation of fitness (replicative ability) changes, as well as changes in the viral sequences during evolution under different selective pressures. We will further analyze the dynamics of these populations by testing the relative contribution of specific mutations to particular phenotypes. It has been well established that RNA viruses have very high mutation frequencies. Such mutation rates, and other sources of genetic variability, such as recombination and reassortment, lead to extremely heterogeneous populations termed "quasispecies." Extreme heterogeneity allow virus populations to adapt and evolve rapidly, although environmental factors can promote genetic stability. For instance, among human pathogens, HIV1, poliovirus, human influenza A virus, hepatitis C virus, hepatitis B virus (which replicates through RNA templates), have all exhibited a great capability for rapid evolution and adaptation in infected patients. In other instances, such in alphaviruses, remarkable genetic stability can be achieved, and although there are a number of hypothesis explaining stasis, very little experimental support has been provided. The fact that no RNA virus has been effectively controlled so far, demonstrates the need to know how these biological entities face new selective pressures. There is also an overall lack of data concerning the population dynamics of viral quasispecies, which is critical for understanding how viruses respond to environmental challenge. Vaccination, immune therapy, and antiviral drug treatments are likely to alter the interactions among variants in a population, but we do not know the laws governing such interactions. This proposal will continue the work developed during the past few years regarding RNA virus population dynamics. Because of the great complexity of quasispecies, there is an unavoidable level of evolutionary indeterminacy, but we aim to unravel and clarify general basic principles of population genetics. Our work, and that of others, has shown that several principles of evolutionary biology apply to RNA viruses, and this knowledge has opened new ways to design antiviral strategies. We will continue applying experimental approaches that include accurate fitness determinations, sequencing, and manipulation of viral genomes to extend our understanding of virus stability and extinction, and how internal factors (phenotypic flexibility) and external factors (environment) affect genetic change. Specifically, the proposal will address the effect of antibody response and of replication in insect vectors, on virus survival and adaptation.